Modified unF-13 protein and gene

ABSTRACT

A unf. 13 protein and gene encoding it are disclosed which confer sensitivity to B. maydis T toxin and the insecticide methomyl, in cells carrying the gene and expressing the protein. Toxin sensitivity domains of the protein have been identified wherein a modification yields a toxin-insensitive product.

INTRODUCTION

This is a continuation of application Ser. No. 07/342,119, filed Apr.24, 1989, now abandoned, which is a continuation-in-part of U.S. patentapplication Ser. No. 144,557, filed Jan. 14, 1988, now abandoned.

The invention relates to plant pathology, especially to cytoplasmic malesterility (cms) in maize and to pathotoxin specificity in certain cmsmaize lines.

BACKGROUND AND PRIOR ART

Maize (Zea mays L.) plants carrying the Texas male-sterile cytoplasm(cms-T) are particularly susceptible to the fungal pathogen Bipolaris(Helminthosporium) maydis, race T, the causative agent of Southern CornLeaf Blight (Hooker, A. L. et al. (1970) Plant Dis. Rep. 54:708). Ahost-specific pathotoxin (BmT-toxin) isolated from the fungusspecifically alters membrane permeabilities of mitochondria from cms-Tmaize. For structure of BmT and related toxins see Frantzen, K. A. etal. (1987) Plant Physiol. 83:863. The carbamate insecticide, methomyl,although structurally unrelated, mimics the BmT-toxin effects (Humaydan,H. S. and Scott, E. W. (1977) Hortic. Sci. 12:312). For structure ofmethomyl and analogs toxic to cms-T maize mitochondria, Arandaet al(1987) see Phytochem. 26:1909. Mitochondria from maize plants containingeither the S or C male-sterile or normal (male-fertile) cytoplasms areunaffected by the BmT-toxin or methomyl. The site of toxin and methomylaction is believed to be at the inner mitochondrial membrane. Inresponse to BmT-toxin or methomyl, cms-T mitochondria exhibit rapidswelling, uncoupling of oxidative phosphorylation, inhibition ofmalate-driven respiration and leakage of small molecules such as NAD⁺and Ca⁺⁺ (Miller, R. J. and Koeppe, D. E. (1971) Science 173:67; Koeppe,D. E. et al. (1978) 201:1227; Berville, A. et al. (1984) Plant Physiol.76:508; Klein, R. R. and Koeppe, D. E. (1985) Plant Physiol. 7.7:912;Holden, M. J. and Sze, H. (1984) Plant Physiol. 75:235).

A strict correlation exists between susceptibility to the B. maydispathotoxin and the cytoplasmic male-sterility (cms) trait in maizeplants containing the T cytoplasm. Both traits are maternally inheritedand attempts to separate the two effects have been unsuccessful.Regeneration of cms-T maize callus from tissue cultures both with andwithout BmT-toxin selection has given rise to revertant plants that arenot only resistant to the BmT-toxin, but are also male fertile; nostable revertants have been obtained that are male sterile and toxinresistant or male fertile and toxin sensitive (Brettell, R. I. S. et al.(1979) Maydica 24:203; Umbeck, P. F. and Gengenbach, B. G. (1983) CropSci. 23:584).

A possible explanation for the simultaneous reversion of the two traitsis that a single locus of extranuclear origin encodes both phenotypicalternatives, i.e. male fertility and toxin resistance versus malesterility and susceptibility.

A mitochondrial gene unique to T-cytoplasm of maize has been isolatedand characterized (Dewey, R. F. et al. (1986) Cell 44:439). Designatedurf13-T, it encodes a 13 kd protein associated with the cms trait. Thenuclear fertility restorer gene Rf1 alters the transcript of urf13-T,resulting in a significant decrease in abundance of the 13 kd protein.Also, in cms-T plants that have reverted to male fertility and B. maydisresistance, the urf13-T reading frame has been found to be eitheraltered or the gene completely deleted. The urf13-T gene has beensequenced and the amino acid sequence of the protein it encodes has beendeduced (Dewey, R. E. et al. (1987) Proc. Natl. Acad. Sci. USA 84:5374).

Preincubation of cms-T maize mitochondria with dicyclohexylcarbodiimide(DCCD), a reagent that preferentially binds covalently to carboxylgroups in hydrophobic regions of proteins, confers protection againstthe effects of BmT-toxin (Bouthyette, P-Y. et al. (1985) J. Exp. Bot.36:511; Holden, M. J. and Sze, H. (1987) in Plant Mitochondria,Structural, Functional and Physiological Aspects, A. L. Moore and R. B.Beechey, (eds.) Plenum Press, New York, pp. 305-308). Pretreatment ofmitochondria with 4 through 15 μM DCCD prevents toxin-induced inhibitionof malate-dependent oxidation, dissipation of the membrane potential,and leakage of accumulated calcium. Preincubation with the water solublecarbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, does notprotect cms-T mitochondria from toxin action, suggesting that DCCDmodifies a component situated in a hydrophobic environment.

SUMMARY OF THE INVENTION

The invention is based on the discovery that urf13-T can be expressed ina bacterial host and that bacteria transformed by expressing the urf13-Tgene are rendered susceptible to BmT toxin and to methomyl. Furthermore,domains of the protein have been identified that are responsible forconferring toxin sensitivity on the host cells. Modification of one ormore of these domains abolishes the specific toxic effects of BmT toxinand of methomyl. The findings provide a basis for a simple assay fortoxin and a screening method for detecting toxin. Expression of theurf13-T gene in bacteria provides a means for destroying those bacteria,if so desired, at any time, by exposing them to BmT toxin or to methomylor to any of the toxic analogs thereof. Such a trait can be used, forexample, if the bacterial strain has been released into an undesiredlocation, where it can be destroyed without harming other organisms.Risk management of bacteria released into the environment is enhanced byincorporating the urf13-T gene into the released strain. A method isalso provided for assessing the effects on toxin sensitivity of specificgene modifications. Modified urf13-T genes can be introduced into plantsand expressed to create sterility that is not accompanied by toxinsensitivity. The modified genes and the modified proteins they encodeare useful for elucidating the structural domains necessary forproducing sterility of male reproductive tissues.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the amino acid sequence of the urf13-T protein (singleletter amino acid abbreviations) superimposed over a hydropathy profileconstructed according to the values of Kyle, J. et al. (1982) J. Mol.Biol. 157:105. Hydrophobic regions have positive values and hydrophilicregions have negative values. The boxed sequence represents amino acids2 through 11, one of the toxin-sensitivity domains. Amino acids incapitals designate the immunogen of the PEP 13 antibody described byDewey, R. E. et al. (1987).

FIG. 2 shows the results of four O₂ electrode measurements of O₂ uptakeby E. coli cells. O₂ uptake on the vertical dimension and time on thehorizontal axis are shown to scales in the inset. Arrows labeled "Toxin"and "Methomyl" show times of addition of BmT toxin and methomyl,respectively. Trace A shows respiration of E. coli JM109 transformedwith pATH3 (lacking an urf13-T insert). B and C show results with JM109transformed with urf13-T. Trace D shows results with JM109 transformedwith pJG13-T, a vector having an inserted TI-urf13-T gene deletion inthe region coding for amino acids 2 through 11.

FIG. 3 Recording of absorbance (A₅₂₀) of spheroplast suspensionsfollowing treatment with BmT (toxin) and methomyl. The scale ofabsorbance (vertical dimension) and time (horizontal) dimension is shownin the inset. Trace A: pATH3-transformed E. coli spheroplasts. Traces Band C are E. coli expressing urf13-T, spheroplasts treated with toxinand methomyl, respectively, at times indicated by the arrows.

FIG. 4 shows leakage of preloaded cells following treatment with BmTtoxin. The data are normalized, 100% uptake is defined as uptake of 0.86Rb after 30 minutes by control cells. A decrease in uptake indicates ionleakage by the cells.

DETAILED DESCRIPTION OF THE INVENTION

The nucleotide sequence of urf13-T has been published (Dewey, R. E. etal. (1986) Cell 44:439). The deduced amino acid sequence of the proteinencoded by urf13-T is shown in Table 1. In higher plant mitochondria,CGG codons are believed to designate tryptophan rather than arginine aspredicted by the universal code (Fox, T. D. and Leaver, C. J. (1981)Cell 26:315). urf13-T and pATH13-T contain a single CGG codon,corresponding to amino acid position 87 (Dewey, R. E. et al. (1986) Cell44:439).

The urf13-T gene can be cloned in a plasmid expression vector andexpressed in a microbial host. Surprisingly, the transformed hostexpressing urf13-T is rendered susceptible to BmT toxin and methomyl,although in the absence of these agents, the host appears to grownormally and respires normally. In the presence of toxin, respiration(O₂ consumption) is completely inhibited by toxin. The time required tocompletely inhibit respiration varies with toxin dose: lower doses oftoxin require longer times to inhibit respiration completely. Toxin alsocauses swelling of spheroplasts of the host organism, reflectingpermeabilized membranes. An alternative assay for membranepermeabilization is the monitoring of ion leakage, after exposure totoxin, from cells preloaded with ⁸⁶ Rb (a radioactive substitute forK⁺).

The urf13-T protein is found associated with the microbial membrane.Still more surprising is the fact that specific gene alterations can bemade that result in expression of a modified urf13-T protein that nolonger confers BmT or methomyl sensitivity to the microbial hostexpressing it, despite the fact that such alterations do not affect theassociation of the modified protein with the host membrane. Experimentsdisclosing these features are described in Examples 1, 2, 3 and 4.

The findings provide a basis for a method of detecting and measuring BmTtoxin and other compounds such as methomyl having toxin-like specificityfor plants having the T cytoplasm. The method involves culturing amicroorganism transformed by and expressing the urf13-T gene, dividingthe culture into samples and treating the samples respectively with thematerial to be tested, a sample of known toxicity and a control known tobe nontoxic, and measuring O₂ consumption and/or ion leakage in eachsample. By comparing the time required to reach a defined level ofinhibition for example, complete (100%) inhibition or 50% inhibition,over a range of known toxin concentrations, a standard curve can bedeveloped against which the amount of toxin in the test sample can bequantified. Less than 7.8 ng/ml of BmT toxin can be readily measured.Nonspecific (unrelated to interaction with urf13-T protein) inhibitionof respiration of test samples can be corrected for by use of a controltest using untransformed cells, grown to approximately the same density.Rates of ⁸⁶ Rb leakage from test samples can be compared to rates ofleakage from cells transformed with a plasmid that does not contain theurf13-T gene insert. Typically, such control cells show no detectableleakage for at least forty minutes following exposure to toxin ormethomyl. These assays may be used qualitatively as a method forscreening for BmT toxin in unknown samples. The methods are thereforeuseful diagnostics to identify the presence of B maydis race T in fieldsamples. Other parameters of the assays can be varied and optimized asdesired according to principles understood by those of ordinary skill inthe art. In particular, the choice of host microorganism and expressionsystem can influence the sensitivity of the assays. E. coli, used in theexample, is very convenient because it is easy and safe to culture andthere are many vectors and expression systems available for use with E.coli. Other organisms may present other advantages.. The resultsdescribed herein demonstrate that the urf13-T protein becomes associatedwith membranes in organisms as diverse as maize and E. coli. Inprinciple, any transformable organism capable of aerobic respiration canserve as host for the urf13-T gene which, if expressed to a sufficientlevel, will render the organism susceptible to inhibition of respirationand energy-dependent ion uptake by exposure to BmT, methomyl and othercompounds having similar toxic specificity.

The urf13-T gene provides a useful "silver bullet" for microorganismstrains that must be inactivated swiftly and selectively. Bacteria thatexpress the urf13-T gene introduced into the environment can beselectively destroyed by treatment with methomyl or by application ofBmT. Methomyl has been used commercially as an insecticide under thename "Lannate" (a trademark of DuPont, Wilmington, Del.). While thesecompounds have a nonspecific toxicity at higher doses, both arespecifically toxic to microorganisms expressing urf13-T at lower doses.Analogs of methomyl have been found that are of comparable toxicity toBmT (Aranda, G. et al. (1987) Phytochem. 26:1909). For brevity, BmT,methomyl and their analogs having similar toxic specificity will betermed simply "toxin" herein. The ability to selectively inactivate agiven microorganism can be used to advantage in laboratory work as well.For example, where an organism is considered hazardous, incorporatingexpressible urf13-T gene provides a failsafe means of biologicalcontainment.

The results described herein provide a method for evaluating the effectson toxin sensitivity of various modifications of the urf13-T gene. Asdescribed in Examples 1 and 4, various modified urf13-T genes have beenprepared and expressed in a microorganism. Modifications at certainamino acid residues result in loss of toxin sensitivity, while manymodifications have no effect. The results demonstrate that specificregions of the urf13-T protein are required for one of its functionalproperties, the ability to confer toxin sensitivity on organisms thatexpress it. Such specific regions are termed herein toxin sensitivitydomains. Modification within a toxin sensitivity domain resulting inloss of toxin sensitivity is termed a sensitivity-loss modification.Modification of the urf13-T gene such that one or more toxin sensitivitydomains is deleted or altered in sequence can result, upon expression,in synthesis of a modified urf13-T protein having a sensitivity-lossmodification, i.e., that fails to confer toxin sensitivity on theorganism expressing it. It is important that, while more than one toxinsensitivity domain exists in the urf13,T protein, a total loss of thetoxin sensitivity function is achievable by a modification of a singledomain. It is possible that not every modification within a toxinsensitivity domain will result in a sensitivity-loss modification. Atoxin-sensitivity domain is variable in size, with an upper limit ofabout 10% or less of urf13-T, or coding for not more than 12 aminoacids. The urf13-T gene can be modified at more than one toxinsensitivity domain to reduce the probability of subsequent reversion totoxin sensitivity in a host organism. The probability of two independentreversions occurring in a doubly modified protein is the product of theindividual probabilities of reversion at each of the modified domains.

The method of measuring the effects of specific modifications of theurf13-T gene includes cloning the modified gene in a suitable plasmidexpression vector, expressing the modified gene in a host microorganismand measuring the O₂ consumption and/or ion leakage of the hostmicroorganism in the presence and absence of BmT or methomyl or toxinanalogs thereof. The modifications may be nucleotide base changes,sequence deletions, sequence insertions, frame shifts or the like asunderstood by those skilled in the art. The techniques for introducingsuch modifications are known to those of ordinary skill in the art.

A modified urf13-T protein that lacks the function of conferring toxinsensitivity to a host organism that expresses it is termed atoxin-insensitive urf13-T protein herein abbreviated TI-urf13-T protein.Its modified gene is hence a TI-urf13-T gene. The designation,TI-urf13-T gene, specifically refers to a gene bearing asensitivity-loss modification. The fact that discrete toxin sensitivitydomains have been discovered indicates that the loss of toxinsensitivity is not the result of a general, nonspecific disruption ofprotein structure or conformation. TI-urf13-T proteins continue to belocalized in the microorganism host cell membrane. Loss of toxinsensitivity therefore does not necessarily lead to loss of otherfunctions. A modified urf13-T gene that yields toxin-sensitivity isdeemed equivalent to unmodified urf13-T and is not separatelydesignated, but simply referred to as an urf13-T gene herein.

A TI-urf13-T gene can be used to develop host cells and mitochondriathat lack toxin sensitivity but otherwise possess the characteristicsthat result in male sterility, under appropriate developmentalconditions. The male sterility phenotype is not manifested bycurrently-known characteristics of single cells. It is not known whetherthe urf13-T protein causes male sterility. Although it is uniquelyexpressed by T-mitochondria, its origin appears to be the result ofseveral gene rearrangements (Dewey et al. (1985), Dewey et al. (1987)).Other effects that are the consequence of these rearrangements may beresponsible for, or contribute to, the Cms phenotype. Furtherunderstanding of the interaction of the urf13-T gene and its expressionproduct with the components of the respiratory system of mitochondria ormicroorganisms will provide a basis for utilizing TI-urf13-T in plantsto produce toxin-insensitive male sterility.

Using the disclosed method for evaluating the ability to confer toxinsensitivity makes it possible to map the urf13-T gene to locate domainsresponsible for toxin sensitivity. The gene can be modified by any of avariety of mutagenic techniques known in the art. Site-specificmutagenesis is a preferred method, using a series of mismatcholigonucleotide primers for introducing nucleotide sequence variationsat any desired locus within the gene. The exact sequence change producedby such means can be identified by nucleotide sequence determination inthe region affected by the change. A modified gene can be tested forfunctional effect of the modification by cloning the gene in anexpression vector, as previously disclosed herein, or using anexpression system known in the art, and expressing the modified gene ina microorganism transformed thereby. If the transformed microorganismexpressing the modified gene is sensitive to toxin at a level comparableto the same organism expressing unmodified urf13-T, the modified gene isclassified as "sensitive," indicating that the modified proteinexpressed thereby confers toxin sensitivity on the microorganismexpressing it. If the microorganism is not toxin sensitive (aftercontrol experiments demonstrate that the modified protein is made andlocalized in the microorganism membrane) the modified gene is classed as"insensitive." Membrane localization is measured by binding ofdicyclohexylcarbodiimide (DCCD). The fact that DCCD only binds tospecific residues of proteins in hydrophobic environments provides aconvenient assay for the intracellular location of proteins encoded bymodified urf-13-T genes. The use of ¹⁴ C-labeled DCCD provides astraightforward assay for monitoring the binding of DCCD to the protein.Only when the protein is inserted into a membrane will the labeled DCCDbecome covalently bound to it.

The described procedure has been used to identify three domains whereinmodifications lead to insensitive (TI-urf13-T) genes: 1) Deletion ofamino acids 2 through 11 results in insensitivity. There is therefore atoxin sensitivity domain within the sequence of amino acids 2 through11. 2) A toxin sensitivity domain beginning at amino acid 83 exists.Deletion of carboxy-terminal amino acids from 84 to the carboxy end hasno effect on toxin sensitivity. However, conversion of the codon foramino acid 83 to a termination (stop) codon results in atoxin-insensitive protein. Thus, a critical length for toxin sensitivityis defined by amino acid 83. Amino acid 83 is required fortoxin-sensitivity under certain conditions. The domain comprising aminoacid 83 extends from there toward the carboxy terminus of the protein.3) A third toxin sensitivity domain comprising amino acid 39 has alsobeen discovered. Mutation of the coding sequence for the amino acid atposition 39 (an aspartic acid residue in the wild-type version of theprotein) to code for either histidine glutamic acid, valine or alanineresults in a toxin-insensitive protein. The conversion totoxin-insensitivity by the conservative replacement with glutamic acid(i.e., an acidic residue replacing an acidic residue) is surprising, andit indicates that the presence of an aspartic acid residue at position39 in the 115 amino acid protein is essential for conferring toxinsensitivity. All domains are independent in the sense that asensitivity-loss modification in any domain alone is sufficient toconfer toxin insensitivity.

It will be apparent to those of ordinary skill in the art thatadditional mutations can be tested and a complete map of all toxinsensitivity domains obtained if desired, so that those of ordinary skillcan generate TI-urf13-T genes following the teachings herein, withoutundue experimentation. Further, it will be apparent that one can findthe minimum modifications, perhaps even single base changes, within eachtoxin sensitivity domain that result in toxin insensitivity. One canalso construct a doubly modified gene that has modifications in morethan one toxin sensitivity domain. The probability of reversion to toxinsensitivity is thereby reduced by several orders of magnitude.

Expression of a TI-urf13-T gene leads to synthesis of a TI-urf13-Tprotein. Expression vectors are well-known in the art. Choice ofexpression vector is a matter of the exercise of ordinary skill in theart, taking into consideration the characteristics of the expressionsystem and the desired means of inducing protein synthesis. Two vectorsfor expression in E. coli have been used in the experiments described inthe examples. Using the pATH vector, the urf13-T gene or its modifiedcounterparts is expressed under the trpE promoter control. Anotherpreferred vector termed PLC236, uses the lambda P_(L) promoter,regulated by a temperature sensitivity repressor (pPLC36, Remaut, E.(1981) Gene 15:81). Use of the P_(L) vector is recommended forexpression in E. coli because the induction is accomplished by atemperature shift. Publicly-available vectors are also suitable forexpressing urf13-T, for example, the pPOP series, the pLG series (200,338, 339 and 400) the pKT series (279,280, 287) and lambda gt11, allavailable from the American Type Culture Collection, 12301 ParklawnDrive, Rockville, Md. USA.

Modified TI-urf13-T proteins are synthesized by cells expressing themodified gene. The proteins can be isolated and purified if desiredusing techniques previously disclosed by Dewey et al. (1987). Thereference disclosed antibodies specific for urf13-T protein. One suchantibody was raised against an oligopeptide composed of amino acids 92through 106, designated PEP 13 therein. Another antigenic peptide hasbeen disclosed by Wise, R. P. et al. (1987) Plant Mol. Biol. 9:121,together with an antibody thereto. Designated PEP 17, the epitope iscomposed of the urf13-T sequence of amino acids 32-48. ModifiedTI-urf13-T peptides that possess either sequence, for examples, peptidesmodified in a toxin-sensitivity domain of amino acids 2 through 11 reactwith either antibody. The antibody can be used to assay such TI-urf13-Tproteins or it can be used for purification by affinity chromatography.Similar antibodies can be raised by those of ordinary skill in the artfor assaying and purifying the TI-urf13-T protein modified in the aminoacids 83 through 85 domain. While the disclosed TI-urf13-T proteinmodified in domain 83 through 85 lacks amino acids 83 through 115, otherproteins modified in the same domain can be full length and can beassayed by the same PEP 13 antibody disclosed previously by Dewey et al.(1987).

Both TI-urf13-T and urf13-T proteins can also be identified throughtheir binding to ¹⁴ C-labeled DCCD. Membrane preparations containingthese proteins are incubated with the labeled DCCD, and the proteins aresubsequently visualized by fluorography, an exercise of ordinary skillin the art.

The invention is further disclosed in the following examples. Theexamples show how the urf13-T gene was cloned in an expression vector,introduced into E. coli and expressed in E. coli. The effects of BmT andmethomyl on transformed E. Coli were tested. Modified urf13-T genes wereconstructed and their activity in a toxin-sensitivity assay tested.Toxin-sensitivity domains were identified, as described. Because thephysiological effect of urf13-T protein in E. coli is the same as inmaize with respect to toxin sensitivity, it is evident that anymicroorganism with a respiratory system will respond similarly.Therefore the procedure described in the examples can be repeated forother microorganisms without difficulty by those of ordinary skill inthe art. Modifications that optimize the procedures for other organisms,choice of media, growth conditions, choice of expression vector,transformation protocols and the like are readily available andunderstood by the person of ordinary skill.

Except as noted hereafter, standard techniques for cloning, DNAisolation, amplification and purification, for enzymatic reactionsinvolving DNA ligase, DNA polymerase, restriction endonucleases and thelike, and various separation techniques, are those known and commonlyemployed by those skilled in the art. A number of standard techniquesare described in: Maniatis et al. (1982) Molecular Cloning, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y.; Wu (ed.) (1979) MethodsEnzymol. 68: Wu et al. (eds.) (1983) Methods Enzymol. 100 and 101;Grossman and Moldave (eds.) Methods Enzymol. 65: Miller (ed.) (1972)Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y.; Old and Primrose (1981) Principles of GeneManipulation, University of California Press, Berkeley; Schleif andWensink (1982) Practical Methods in Molecular Biology; Glover (ed.)(1985) DNA Cloning Vol. I and II, IRL Press, Oxford, UK; Hames andHiggins (eds.) (1985) Nucleic Acid Hybridisation, IRL Press, Oxford, UK;and Setlow and Hollander 1(1979) Genetic Engineering: Principles andMethods, Vols. 1-4, Plenum Press, New York, which are incorporated byreference herein. Abbreviations, where employed, are those deemedstandard in the field and commonly used in professional journals such asthose cited herein.

EXAMPLES Example 1

The entire urf13-T gene was cloned into the plasmid vector pATH 3 andexpressed in E. coli strain JM109 (Yanish-Perran, C. et al. (1985) Gene33:103) ATCC accession no. 53323, as described.

The pATH13-T plasmid was constructed using the pATH3 expression vector,the urf13-T gene of cms-T mitochondria, and two complementary syntheticoligonucleotides. The urf13-T gene contains two BclI restriction sites,one of which cuts at the putative initiator methionine codon, Digestionof the clone containing urf13-T with the restriction endonucleaseHindIII, followed by a partial digestion with BclI enabled us to obtaina fragment that contained the entire urf13-T gene except for the firsttwo nucleotides of the ATG initiation codon (plus approximately 450 basepairs of 3' flanking region). The pATH 3 plasmid was linearized bycleavage of the EcoRI and HindIII sites located within the polycloningregion of the vector. Two complementary oligonucleotides correspondingto the sequences 5'-AATTCTGGAGGAAAAAATTAT-3' (top strand) and5'-GATCATAATTTTTTCCTCCAG-3' (bottom strand) were annealed to yield afragment with EcoRI and BclI sticky ends. The linear pATH 3 vector andthe BclI-HindIII fragment containing urf13-T were joined by ligation ofthe EcoRI end of pATH 3 to the EcoRI end of the oligomer fragment,ligation of the BclI end of the oligomer fragment to the BclI end of theurf13-T sequence, and ligation of the HindIII end of the urf13-Tfragment to the HindIII end of pATH 3. The oligonucleotides wereconstructed to provide a procaryotic ribosome binding site and toreconstitute the initiator ATG codon of urf13-T. The integrity of thepATH13-T construct was verified by nucleotide sequence analysis. Theoligonucleotides were prepared with the Applied Biosystems 380A DNASynthesizer according to the manufacturer's instructions. Cloningprocedures were conducted as described by T. Maniatis, E. F. Fritsch, J.Sambrook, in Molecular Cloning, A Laboratory Manual (Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., 1982). The structure of the pATH 3expression vector and conditions of transcriptional induction of the trppromoter were according to Spindler, K. R. Rosser, D. S. E. Berk, A. J.(1984) J. Virol. 49:132, with modifications described by T. J. Koerner(personal communication). Optimal expression of the urf13-T in thepATH13-T vector was obtained in an M9 medium with amino acids minustryptophan, glucose and thiamine. Although the pATH 3 expression vectorwas designed to enable synthesis of trp E fusion proteins (see, e.g.,Dickmani et al. (1985) J. Biol. Chem. 260:1513), the pATH13-T plasmidwas constructed so that the urf13-T sequence was out of frame with thetrpE reading frame allowing production of an unfused 13 kD protein.

The pATH 3--urf13-T construct, designated pATH13-T is transcriptionallyregulated by the trpE promoter of E. coli and produces a 13 kD proteinupon induction. This polypeptide co-migrates electrophoretically withthe mitochondrial urf13-T protein product of cms-T maize onSDS-polyacrylamide gels as determined by protein blot analysis.Nucleotide sequence analysis confirmed that the urf13-T reading frame inpATH13-T was identical with the maize reading frame (data not shown).Although identical in nucleotide sequence, a difference in amino acidcomposition of the pATH13-T and urf13-T proteins may exist due to apossible difference in codon usage.

A modified urf13-T gene that produces a form of the 13 kD proteinmissing amino acids 2 through 11 was also constructed and expressed inE. coli using the plasmid vector pJG200.

A BclI-BglII fragment (positions 1248 to 1723 according to Dewey et al.containing all of the urf! 3-T reading frame except for the first 11codons (along with approximately 150 base pairs of 3' flanking sequence)was ligated into the BamHI site of the expression vector pJG200. Sinceplasmid pJG200 utilizes the initiator methionine codon of the lambda crogene, the resulting construct (pJG13-T) encoded all of the urf13-Treading frame except codons 2 through 11. The pGJG200 vector andconditions for induction of transcription are described by Germino, J.et al. (1983) (Proc. Natl. Acad. Sci. USA 80:6848. and Germino, J. andBastia, D. (1984) Proc. Natl. Acad. Sci. USA 81:4692.

The truncated urf13-T - pJG200 construct, designated pJG13-T, is underthermoinducible control of the cI857 thermolabile repressor and Prpromoter of phage lambda. E- coli cells containing plasmid pJG13-Tabundantly expressed the modified form of the 13 kD protein aftertemperature induction at 42° C. Although the pJG13-T protein product was10 amino acids shorter than the 13 kD protein produced by the pATH13-Tplasmid or the urf13-T gene of cms-T mitochondria, no difference inelectrophoretic mobility was detected by protein blotting.

The truncated protein, like the intact 13 kD polypeptide, is localizedin the membrane of E. coli (data not shown).

Another expression vector, pLC236 (Remaut, E. (1981)) has been usedsuccessfully for expressing the urf13-T gene in modifications thereof.Expression is controlled by the lambda P_(L) promoter and the promoteris thermoinducible. Use of pLC236 is deemed the best of the threevectors exemplified, because of the high expression levels and ease ofinduction. After introducing mutations in the urf13-T gene the mutantsequences (attached to prokaryotic translation signals) were insertedinto the pLC236 vector and transformed into an E. coli host carrying thecI857 gene that produces a temperature-sensitive repressor protein.

Example 2

The effects have been measured of the BmT-toxin and methomyl onrespiration in E. coli cells containing the pATH13-T and pJG13-Tplasmids that have been induced to express the normal and truncated 13kD proteins, respectively.

Additions of 780 ng/ml BmT-toxin and/or 4 mM methomyl were made to E.coli cultures expressing the plasmids pATH3, pATH13-T, and pJG13-T. Thereaction medium contained 42 mM Na₂ HPO₄, 22 mM KH₂ PO₄, 8.5 mM NaCl, 19mM NH₄ Cl, 10 mM glucose and from 150 to 200 μg E. coli protein. O₂consumption was measured polarographically with a Clark oxygenelectrode. Respiration rates were expressed as nmol O₂ consumed/min/mgE. coli protein.

O₂ consumption was completely inhibited by the addition of 780 ng/mlBmT-toxin or 4mM methomyl in cells producing the complete 13 kD protein.Respiration was not altered by toxin or methomyl in control cellstransformed with the pATH 3 vector containing no insert, or in the cellsproducing the truncated version of the 13 kD protein.

The time required to completely inhibit respiration after toxin additionin E. coli cells was dependent on the concentration of the toxin.Although O₂ consumption was completely inhibited at toxin concentrationsof 7.8 ng/ml, eight to nine minutes were required for full inhibition.In contrast, complete inhibition was achieved after approximately oneminute with 780 ng/ml of toxin (FIG. 1B).

Example 3

In cms-T mitochondria, BmT-toxin and methomyl cause a rapid decrease inA₅₂₀ that has been interpreted as mitochondrial swelling (Miller, R. J.et al. (1971) Science 173:67; Koeppe, D. E. et al. (1978) Science210:1227; Berville, A. et al. (1984) Plant Physiol. 76:508; Klein, R. R.et al. (1985) Plant Physiol. 77.:912). To determine whether E. colicells synthesizing the 13 kD protein showed similar effects, changes inabsorbance were recorded for spheroplasts from pATH13-T induced E. colicells in the presence of toxin and methomyl.

E. coli cells expressing the plasmids pATH 3 and pATH13-T were treatedwith 780 ng/ml toxin and 4mM methomyl. E. coli spheroplasts wereprepared as previously outlined (Burstein, C. et al. (1979) Eur. J.Biochem. 9.4:387). Absorbance measurements were madespectrophotometrically in a medium containing 10mM Tris-HCl pH 8.0, 30%sucrose, 10 mMEDTA, 10 mM glucose, and from 0.45 to 0.6 mg of E. coliprotein.

BmT-toxin and methomyl induced dramatic swelling in E. coli spheroplaststhat produced the 13 kD polypeptide; no effect was seen withspheroplasts containing the pATH 3 control plasmid. In accord with therespiration results, E. coli spheroplasts producing the truncated 13 kDprotein from plasmid pJG13-T showed no stimulation in swelling after theaddition of toxin or methomyl.

To determine the effect of the BmT-toxin on E. coli growth, the A₅₅₀ ofPATH13-T induced cell cultures was monitored for several hours both inthe presence and absence of toxin. No growth was detected in E. colicultures (log phase) expressing the 13 kD polypeptide during a six hourperiod after addition of BmT-toxin (780 ng/ml), whereas the same cellswithout toxin exhibited growth rates similar to cells containing thepATH 3 control plasmid.

Example 4

The effects of BmT toxin on ion-leakage in E. coli cells expressingurf13-T and TIurf13-T genes have been measured.

Four different cultures of E. coli were used: 1) "Control," containing apLC236 plasmid with no insert, 2) "T-urf13," containing a pLC236 plasmidwith the full-length urf13-T gene inserted, 3) "92 aa," containing apLC236 plasmid with a truncated Urf13-T gene coding for the first 92amino acids of the 13 KDa urf13-T protein, and 4) "82aa," containing apLC236 plasmid with a truncated urf13-T gene coding for the first 82amino acids of the urf13-T protein.

All four cultures were incubated for 30 minutes in the presence of ⁸⁶ Rbto allow energy-dependent uptake of the isotope (as described in Braunet al. (1989) in The Molecular Basis of Plant Development, Vol 92:79-85.All four cultures responded similarly, accumulating comparable amountsof ⁸⁶ Rb. BmT toxin, at a concentration of 1 μM, was added to eachculture after 30 minutes. The cultures expressing the full-lengthurf13-T protein or the 92 amino acid (truncated) urf13-T protein wereseverely affected by addition of the toxin, showing rapid loss of the ⁸⁶Rb ions from the cells within the first minute of exposure to toxin. Incontrast, the control culture and the culture expressing the 82 aminoacid (truncated) urf13-T protein showed no leakage of ions from thecells upon exposure to toxin.

Example 5

Various modifications of the urf13-T gene have been tested for theireffects on the toxin sensitivity conferred by expression of the urf13-Tgene itself. The modifications were made using known techniques ofsite-directed mutagenesis, using mismatch oligonucleotide primers. Theefficiency of recovery of mutants was enhanced using the technique ofKunkel, T. (1985) Proc. Natl. Acad. Sci. USA 82..:488. The nature of thenucleotide sequence change produced in the mutant was confirmed directlyby sequence analysis over the region modified. The modified genes werethen expressed essentially as described in Example 1. Strains expressingthe modified genes were tested for BmT and methomyl sensitivity asdescribed in Example 2. Results are tabulated in Table 1. Where themodification had no effect (produced the same level of toxin sensitivityas unmodified urf13-T) the result was scored as "sensitive." When themodification yielded an organism that lacked toxin sensitivity, it wasscored as "insensitive." Three domains were identified when amodification resulted in a loss of toxin sensitivity. Near the --NH₂end, amino acids 2 through 11 were seen to be essential for toxinsensitivity. The presence of amino acid 83 was also essential for toxinsensitivity. Deletion of the entire --COOH terminus from amino acids 84through 115 had no effect on toxin sensitivity. However, insertion of astop codon at position 83 resulted in toxin insensitivity. An internaldeletion that eliminates the leucine residue at position 83, leading toa protein 114 amino acids in length, results in a toxin-sensitiveprotein. Therefore, a critical length of 83 amino acids is required fortoxin sensitivity. The amino acid at position 39 is also essential fortoxin sensitivity. If the DNA sequence is modified so that the aminoacid at position 39 is histidine, glutamate, valine or alanine, theprotein is toxin-insensitive. Thus, the presence of an aspartate moietyat position 39 is essential to toxin sensitivity of the 115 amino acidurf13-T protein. Modifications that result in a different amino acidbeing present at position 39 in the 115 amino acid protein will producea toxin-insensitive protein.

Additionally, certain mutations at positions 56 or 67, which substituteda basic amino acid (lysine) for an acidic amino acid, dramaticallyaffected the quantity of protein accumulated in the cells. Thisindicates that the introduction of positively charged amino acidsbetween positions 55 and 68 lead to instability of the resultingprotein.

From the foregoing it will be apparent that other modifications thanthose disclosed herein can lead to construction of TI-urf! 3-T genes,and synthesis of other TI-urf13-T proteins having other sorts ofnucleotide sequence modifications than the ones disclosed herein. Suchmodifications can be made within the domains identified above that areessential for toxin sensitivity. Modifications within those domains neednot involve all amino acids within the domain and may indeed requireonly a single amino acid change within the domain to produce the toxininsensitivity phenotype. Furthermore, it will be understood that silentmutations within the toxin sensitivity domains can be made, i.e., thosethat do not result in loss of toxin sensitivity. Instances of silentmutations are shown in Table 1. Furthermore, it will be readily apparentthat the teachings and disclosures herein, as well as other teachingsknown in the art, make further mapping of the urf13-T gene andidentification of other toxin sensitivity domains if they exist, amatter of routine. All such modifications and mutations that yield aTI-urf13-T gene or a TI-urf13-T protein therefore lie within the scopeof the present invention.

                  TABLE 1                                                         ______________________________________                                        The effect of specific amino acid changes in urf13 on                         the sensitivity of E. coli to the T-toxin.                                    Amino                                                                         acid    Original          Mutant     Toxin                                    number(s)                                                                             Residue           Residue    Effect                                   ______________________________________                                        2-11    Ile--Thr--Thr--Phe--Leu--                                                                       deleted     .sub.--insens-                                  Asn--Leu--Pro--Pro--Phe      itive                                    2       Ile               deleted    sens-                                                                         itive                                    2       Ile               Asn        sens-                                                                         itive                                    2       Ile               Ser        sens-                                                                         itive                                    8       Leu               Arg        sens-                                                                         itive                                    8       Leu               His        sens-                                                                         itive                                    9       Pro               Arg        sens-                                                                         itive                                    9       Pro               His        sens-                                                                         itive                                    9       Pro               Leu        sens-                                                                         itive                                    10      Pro               Arg        sens-                                                                         itive                                    10      Pro               His        sens-                                                                         itive                                    10      Pro               Leu        sens-                                                                         itive                                    12      Asp               Glu        sens-                                                                         itive                                    12      Asp               Arg        sens-                                                                         itive                                    12      Asp               Gly        sens-                                                                         itive                                    12      Asp               His        sens-                                                                         itive                                    12      Asp               Ala        sens-                                                                         itive                                    12      Asp               Val        sens-                                                                         itive                                    12; 39  Asp; Asp          Val; Ala    .sub.--insens-                                                               itive                                    12; 39-40                                                                             Asp; Asp--Asp     Gly;        .sub.--insens-                                                    Ala--His   itive                                    12; 40  Asp; Asp          Gly; His   sens-                                                                         itive                                    12; 48  Asp; Glu          Arg; Gln   sens-                                                                         itive                                    12; 67  Asp; Glu          Arg; Gln   sens-                                                                         itive                                    27      Cys               Ser        sens-                                                                         itive                                    27      Cys               Pro        sens-                                                                         itive                                    39      Asp               Glu         .sub.--insens-                                                               itive                                    39      Asp               His         .sub.--insens-                                                               itive                                    39      Asp               Val         .sub.--insens-                                                               itive                                    39-40   Asp--Asp          Ala; His    .sub.--insens-                                                               itive                                    40      Asp               His        sens-                                                                         itive                                    40      Asp               Tyr        sens-                                                                         itive                                    48      Glu               Gln        sens-                                                                         itive                                    48      Glu               Lys        sens-                                                                         itive                                    56      Glu               Gln        sens-                                                                         itive                                    67      Glu               Gln        sens-                                                                         itive                                    72-73-74                                                                              Trp--Leu--Arg     Ser--Gly-    .sub.--insens-                                                   STOP       itive                                    83      Leu               deleted    sens-                                                                         itive                                    83      Leu               STOP        .sub.--insens-                                                               itive                                    84      Pro               STOP       sens-                                                                         itive                                    85      Ile               STOP       sens-                                                                         itive                                    86      Gln               STOP       sens-                                                                         itive                                    88      Asn               STOP       sens-                                                                         itive                                    93      Arg               STOP       sens-                                                                         itive                                    102     Lys               STOP       sens-                                                                         itive                                    ______________________________________                                    

We claim:
 1. A TI-urf 13-T gene, which gene comprises a modification ofthe wild-type urf13-T coding sequence within a toxin-sensitivity domainof a wild-type urf13-T protein, said modification consisting essentiallyof a deletion of the region of said wild-type urfT-13 coding sequenceencoding amino acids 2 through 11 of the wild-type urf13-T protein.
 2. ATI-urf13-T gene, which gene comprises a modification of the wild-typeurf13-T coding sequence within a toxin-sensitivity domain of a wild-typeurf13-T protein, said modification consisting essentially of a deletionof the region of said wild-type urf 13-T coding sequence encoding aminoacids 83 through 115 of the wild-type urf13-T protein.
 3. A TI-urf13-Tgene, which gene comprises a modification of the wild-type urf13-Tcoding sequence within a toxin-sensitivity domain of a wild-type urf13-Tprotein, said modification consisting essentially of a substitution of acodon for an amino acid which is not aspartate at the region of saidwild-type urf13-T coding sequence encoding amino acid 39 of a wild-typeurf13-T protein.
 4. The TI-urf13-T gene of claim 3, wherein saidmodification of the wild-type urf13-T coding sequence within atoxin-sensitivity domain of a wild-type urf13-T protein consistsessentially of a substitution of a codon for an amino acid selected fromthe group consisting of glutamate, histidine, valine and alanine foraspartate at amino acid 39 in said of a wild-type urf13-T protein.
 5. Aplasmid expression vector comprising a TI-urf13-T gene, which genecomprises a modification of the wild-type urf13-T coding sequence withina toxin-sensitivity domain of a wild-type urf13-T protein, saidmodification consisting essentially of a deletion of the region encodingamino acids 2 through 11 of the wild-type urf13-T protein, wherein saidTI-urf13-T gene is expressible in a microorganism under the control of apromoter of said expression vector.
 6. A plasmid expression vectorcomprising a TI-urf13-T gene, which gene comprises a modification of thewild-type urf13-T coding sequence within a toxin-sensitivity domain of awild-type urf13-T protein, said modification consisting essentially of adeletion of the region in said wild-type urf 13-T coding sequenceencoding amino acids 83 through 115 of the wild-type urf13-T protein,and wherein said TI-urf13-T gene is expressible in a microorganism underthe control of a promoter of said expression vector.
 7. A plasmidexpression vector comprising a TI-urf13-T gene, which gene comprises amodification of the wild-type urf13-T coding sequence within atoxin-sensitivity domain of a wild-type urf13-T protein, saidmodification consisting essentially of a substitution of a codonencoding an amino acid which is not aspartate for the codon of saidwild-type urf 13-T coding sequence which encodes amino acid 39 of thewild-type urf13-T protein, wherein said TI-urf13-T gene is expressiblein a microorganism under the control of a promoter of said expressionvector.
 8. The plasmid expression vector of claim 52, wherein saidmodification of the wild-type urf13-T coding sequence within atoxin-sensitivity domain of a wild-type urf13-T protein consistsessentially of a substitution of a codon encoding an amino acid selectedfrom the group consisting of glutamate, histidine, valine and alaninefor the codon of said wild-type urf 13-T coding sequence which encodesamino acid 39 of the wild-type urf13-T protein.